U.S. patent application number 10/258616 was filed with the patent office on 2003-09-11 for sustained release pharmaceutical compositions for the parenteral administration of hydrophilic compounds.
Invention is credited to Autuori, Francesco, Bianchini, Carlo, Bottoni, Giuseppe, Leoni, Flavio, Mascagni, Paolo, Monzani, Valmen, Piccolo, Oreste.
Application Number | 20030171299 10/258616 |
Document ID | / |
Family ID | 11445131 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171299 |
Kind Code |
A1 |
Autuori, Francesco ; et
al. |
September 11, 2003 |
Sustained release pharmaceutical compositions for the parenteral
administration of hydrophilic compounds
Abstract
Stable, biologically compatible pharmaceutical compositions in
the form of water-in-oil microemulsions (w/o), for the sustained
release by parenteral administration of active ingredients which
are hydrophilic or are made hydrophilic by suitable derivatization,
a process for the preparation of said microemulsions and the use
thereof.
Inventors: |
Autuori, Francesco;
(Cinisello Balsamo, IT) ; Bianchini, Carlo;
(Cinisello Balsamo, IT) ; Bottoni, Giuseppe;
(Cinisello Balsamo, IT) ; Leoni, Flavio;
(Cinisello Balsamo, IT) ; Mascagni, Paolo;
(Cinisello Balsamo, IT) ; Monzani, Valmen;
(Cinisello Balsamo, IT) ; Piccolo, Oreste;
(Cinisello Balsamo, IT) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
11445131 |
Appl. No.: |
10/258616 |
Filed: |
November 6, 2002 |
PCT Filed: |
May 23, 2001 |
PCT NO: |
PCT/EP01/05949 |
Current U.S.
Class: |
424/450 ;
514/10.3; 514/10.4; 514/11.1; 514/54; 514/61 |
Current CPC
Class: |
A61P 5/26 20180101; A61P
35/00 20180101; A61P 5/02 20180101; A61K 9/1075 20130101; A61P 5/24
20180101; A61P 5/06 20180101 |
Class at
Publication: |
514/15 ; 514/16;
514/17; 514/54; 514/61 |
International
Class: |
A61K 038/09; A61K
038/08; A61K 031/715 |
Claims
1. A stable, biologically compatible, well tolerated w/o
microemulsion, suitable for the parenteral administration of
biologically active compounds which are hydrophilic or are made
hydrophilic by suitable derivatization, providing the sustained
release of the active compounds contained therein, said
microemulsion consisting of: a) up to 20% of an internal
hydrophilic aqueous phase containing the therapeutically active
hydrophilic compound; b) for 30 to 98% of a hydrophobic external
phase; c) for up to 50% of a surfactant in admixture with a
co-surfactant selected from C.sub.8-C.sub.20 fatty acids;
C.sub.2-C.sub.14 polyhydroxyalkanes; C.sub.2-C.sub.12 alcohols;
esters of lactic acid with a C.sub.2-C.sub.8 alcohol residue.
2. A w/o microemulsion as claimed in claim 1 characterized in that,
after administration to experiment animals, it induces no
persistent ulcerations, any swelling at the injection site being
reversible.
3. A microemulsion as claimed in claim 1 or 2, wherein the
hydrophilic, biologically active compounds are peptides, proteins,
oligo- or polysaccharides.
4. A microemulsion as claimed in claim 3, wherein the biologically
active compound is a peptide.
5. A microemulsion as claimed in claim 4, wherein the biologically
active compound is a LHRH hormone peptide analog.
6. A microemulsion as claimed in claim 5, wherein the biologically
active compound is Leuprolide acetate, Goserelin, Triptorelin,
Nafarelin, Histrelin, Cetrorelix or the corresponding acetates.
7. A microemulsion as claimed in claims 5 or 6, which provides the
sustained release of the active compound for at least 30 days.
8. A microemulsion as claimed in claim 4, wherein the biologically
active compound is Somatostatin, Octreotide or Lanreotide.
9. A formulation as claimed in claim 8, which provides the
sustained release of the peptides for at least 8 days.
10. A microemulsion as claimed in claim 3, wherein the biologically
active compound is an oligo- or polysaccharide.
11. A microemulsion as claimed in claim 10, wherein the
biologically active compound is unfractioned heparin or low
molecular weight heparins.
12. A microemulsion as claimed in claim 1, wherein the surfactant
is selected from the group consisting of: a) natural or synthetic
glycerophospholipids containing residues of C.sub.4-C.sub.20
saturated or unsaturated carboxylic acids, having as phosphoester
moiety a residue of choline, ethanolamine, serine, glycerol; b)
cholesterol; c) sugars C.sub.12-C.sub.20 fatty acid esters; d)
polyoxyethylene sorbitan C.sub.12-C20 fatty acid esters;
13. A microemulsion as claimed in claim 1 wherein the hydrophobic
continuous phase is selected from the following compounds, alone or
in mixture: esters of C.sub.8-C.sub.20 saturated or unsaturated
carboxylic acids with a C.sub.2-C.sub.8 alcohol residue or mono,
di- and triglycerids of C.sub.8-C.sub.20 fatty acids or vegetable
oils suitable for the parenteral administration.
14. A microemulsion as claimed in claim 12, wherein the aqueous
phase containing the biologically active compound has pH between
4.5 and 7.5, preferably between 5 and 7.
15. A microemulsion as claimed in claim 14, wherein pH is adjusted
by addition of natural amino acids.
16. The use of a microemulsion as claimed in the preceding claims,
comprising Leuprolide, Goserelin, Triptorelin, Nafarelin acetate,
Histrelin, Cetrorelix or the corresponding acetates or other LHRH
analogs for the preparation of a medicament for suppressing
testosterone production after single administration for at least 30
days, where testosterone level already decreases 48 h after the
administration.
17. The use of a microemulsion as claimed in the preceding claims,
comprising Octreotide or its analogues for the preparation of a
medicament for suppressing growth hormone production for at least 8
days.
18. The use of a microemulsion as claimed in the preceding claims
containing unfractioned heparin or low molecular weight heparins
for the preparation of medicaments with sustained release after
single administration.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to sustained release
pharmaceutical compositions for the parenteral administration of
active ingredients which are hydrophilic or are made hydrophilic by
suitable derivatization, in the form of stable, biologically
compatible and easily preparable water-in-oil microemulsions (w/o).
More particularly, peptide active ingredients or biologically
active oligo- or polysaccharides, for which protection from the
immediate attack by the hydrolytic enzymes present in living
organisms as well as sustained release in time, to avoid repeated
administrations, are desirable, are advantageously formulated
through said microemulsions. Said formulations for the
therapeutical use can be injected without problems or significant
side effects and are easily prepared industrially, providing a
remarkable technical improvement.
PRIOR ART
[0002] Microemulsions can be generally defined as optically
isotropic systems, not birifrangent under polarized light
observation, transparent, thermodynamically stable, of extremely
reduced size, with droplet diameter ranging from 5 to 200 nm,
produced by dispersion of two immiscible liquids which are
stabilized by the presence of emulsifiers, which modify the
chemical-physical properties of the separation surface between the
two liquids, reducing substantially to zero the interfacial
tension. An oil, water, a surfactant or tenside and optionally of a
co-surfactant o co-tenside should usually be present for
microemulsions to form; the tendency to form a water-in-oil (w/o)
or oil-in-water (o/w) microemulsion depends on the mutual
proportions of aqueous and oily phases as well as on the nature of
the surfactant. In particular, ternary phase diagrams having as
components water, a hydrophobic compound and a mixture of
surfactant and co-surfactant, obtained according to experimental
data, allow to single out the area in which w/o and o/w
microemulsions exist and are stable. For example, Aboofazeli et al
(Int.J.Pharm. 111 (1994) 63-72) studied the capability of various
compounds having co-surfactant action to form w/o microemulsions.
The system studied by the Authors consisted of mixtures of a fatty
acid ester, a 1:1 lecithin-co-surfactant and water in various
ratios. An efficiency scale of the compounds used as co-surfactants
has been defined: primary amines>alcohols>fatty acids.
Furthermore, the efficiency proved to be related with the length of
the alkyl chain of the alcohol and of the respective fatty acid;
hence butanol>pentanol>he- xanol>pentanoic
acid>hexanoic acid. On the basis of these experimental data,
alcohols such as butanol and pentanol and, to a less extent, the
corresponding fatty acids, appear to be the best candidates. The
use of the described compounds allows to prepare stable w/o
microemulsions, but does not ensure the tolerability of said
formulations, particularly for the depot use wherein the
formulation and the subcutaneous or intramuscular tissue remain in
contact for days or even weeks.
[0003] Moreover, literature reports that the co-surfactant to
surfactant ratio is critical; the data reported by Aboofazeli et
al. relate to a 1:1 ratio between the two components. Atwood et al
(Int.J.Pharmacy 84 R5 (1992)) studied the behaviour of
lecithin/water/fatty acid ester/butanol mixtures wherein the
surfactant/co-surfactant ratio is increased from about 1.7 to 3.
The Authors evidenced that the decrease of the co-surfactant amount
in favor of lecithin dramatically restricts the area in which the
presence of a w/o microemulsion is observed, even when using a
remarkably efficient co-surfactant such as butanol.
[0004] Surfactants are generally classified according to an
empirical scale, known as hydrophile-lipophile balance (HLB) which
assignes values ranging from 1 to 40. As a rule, suitable
surfactants for w/o microemulsions have low HLB whereas those
suitable for o/w microemulsions have high HLB. When the interfacial
tension is <2.times.10.sup.-2 dyn/cm, a stable microemulsion can
form. The optional presence of a co-surfactant allows to increase
the interfacial fluidity, as the co-surfactant molecules penetrate
between the surfactant molecules, thereby producing a
dishomogeneous surface film. Co-surfactants can also decrease the
aqueous phase hydrophilia and hence the interfacial tension between
the two phases. In principle, the use of co-surfactants is
advantageous in that they allow to decrease the amount of necessary
surfactant while increasing the stability of the microemulsion;
however, as already mentioned above, it is preferable to limit
their use as they have potential topical toxicity, mainly in case
of contact between the carrier and the subcutaneous or
intramuscular tissues for prolonged times.
[0005] The numerous advantages of the use of a microemulsion as a
carrier for active ingredients are known.
[0006] Microemulsions, under specific conditions of ratios between
the components, can form spontaneously without need for high power
for the preparation thereof; therefore their preparation on an
industrial scale can be easy. Said spontaneous microemulsions are
thermodynamically stable, homogeneous and transparent, so that they
can be monitored by spectroscopical techniques. Microemulsions with
mean diameters <100 nm can be prepared, which can be cold
sterilized by filtration trough 0.22 micron membrane commercial
filters. The microemulsions can allow to administer poorly soluble
or poorly stable drugs.
[0007] Furthermore said systems can undergo phase inversion when an
excess of the dispersed phase is present, or as a consequence of a
change of temperature: this property can affect the bioavailability
of the active ingredient with a mechanism that has not yet been
clarified.
[0008] The w/o microemulsions can control the release of the active
ingredient or protract its stability in physiologic fluids through
protection from the action of hydrolytic enzymes. A number of
reviews on microemulsions exist, for example "Industrial
application of microemulsions" Marcel Dekker Ed. 1997, which in the
chapter "Microemulsions in the Pharmaceutical field: perspectives
and applications" deals with the usefulness thereof in the
pharmaceutical field, and "Handbook of Microemulsion Technology",
Ed. Kumar Mittal (1999) concerning the chemical-physical
aspects.
[0009] The use of w/o microemulsions as vehicles to obtain a
controlled release of active ingredients which are hydrophilic or
are made hydrophilic by suitable derivatization, is reported in the
patent literature.
[0010] In particular, as for biodegradable molecules such as
peptides, the parenteral administration of w/o microemulsions
containing said active ingredients is reported in a study [M. R.
Gasco et al., Int.J.Pharm., 62, 119 (1990)] wherein an LHRH hormone
analog, formulated in a microemulsion consisting of components
considered biocompatible and containing 500 .mu.g/ml of the active
ingredient, administered by single intramuscular injection at doses
of 3 mg/Kg in adult male rats weighing about 200 g, decreased
testosterone plasma levels for a time up to about 30 days after the
injection; said levels were lower than those observed in a second
group of mice treated with repeated injections (one a day for 28
days) at doses of 100 .mu.g/Kg of active ingredient in buffer
solution.
[0011] It should however be noted that the decrease in the
testosterone concentration was not homogeneous in time during
observation, and it became therapeutically effective only 8 days
after the administration.
[0012] This delay in the therapeutical effect is not very
advantageous, particularly in the treatment of prostate tumor which
is known to require testosterone for growing; therefore the faster
the testosterone normal production is stopped, the more effective
the treatment.
[0013] The above mentioned paper shows the effectiveness of a w/o
microemulsion consisting of ethyl oleate (60.5%), water (10.1%),
phosphatidyl choline (18.9%) and caproic acid (10.5%) for the
sustained release of the peptide. The surfactant
(phosphatidylcholine) to co-surfactant (caproic acid) ratio is 1.8.
The components of said microemulsion, individually taken, are
considered biologically compatible by the Authors. No mention is
made of the biocompatibility of the microemulsion as a whole
neither of the condition of the subcutaneous tissue in contact with
the formulation.
[0014] According to the teaching of said paper, a w/o microemulsion
containing Leuprolide acetate as active ingredient with LHRH
activity was prepared, consisting of ethyl oleate (66.9%), water
(9.7%), phosphatidyl choline (19.4%) and a caproic acid/butyric
acid 3/1 mixture (3.9%). According to this paper, the mixture with
butyric acid was used instead of caproic acid alone in order to
minimize the surfactant to co-surfactant ratio, which is in this
case 4.9 instead of 1.8. The in vivo test carried out by
subcutaneous injection of the product in the rat has confirmed the
effectiveness but has also surprisingly shown alarming local
ulcerogenicity and persistent formation of subcutaneous granulomas.
This pharmacologically unacceptable result, notwithstanding the
lower amounts of co-surfactants used, proved that the
biocompatibility of the single components of the microemulsion was
not sufficient to ensure the biocompatibility of the mixture
constituting the microemulsion, when used for the parenteral
administration.
[0015] Similarly, the mixtures disclosed and claimed in various
patents, e.g. WO 94/08610, although providing stable microemulsions
and possible controlled releases of the active ingredient in time,
do not teach to those skilled in the art how to obtain the
sustained release of an hydrophilic active ingredient while
avoiding such side effects. Said microemulsions usually consist, in
fact, of water, an oily component, a surfactant, a co-surfactant,
and optionally electrolytes, in various ratios. Neither
biocompatibility of the microemulsion "in toto" is evaluated, nor
the necessary ratios of mixture components to active ingredient are
indicated, to obtain stability of both the active ingredient and
the microemulsion as well as biocompatibility of the
formulation.
[0016] On the other hand, said patents did not specifically
consider the problems concerning local tolerability connected with
intramuscular (i.m.) and subcutaneous (s.c.) administrations, which
are the most suitable and easy routes for the parenteral
administration.
[0017] An alternative technology already used at the industrial
level for the sustained release of peptides is that described and
claimed in a number of patents, inter alia U.S. Pat. No. 3,976,071,
wherein such release is obtained by the use of bioerodible polymers
in which the active ingredient is inbodyrated. Typical examples of
bioerodible polymers are polymers based on glycolic acid and lactic
acid. The drawback of said technology is that it is relatively
expensive and troublesome compared with the above described
microemulsions, furthermore it requires the use of organic, in
particular chlorinated, solvents during the preparation, which
involves problems in terms of environmental impact and safety of
the pharmaceutical formulation.
[0018] On the other hand, said formulations advantageously cause
not persistent granulomas and do not induce local
ulcerogenicity.
DISCLOSURE OF THE INVENTION
[0019] The present invention aims at providing sustained release
pharmaceutical compositions in the form of stable w/o
microemulsions, which are easy to prepare, can be sterilized, are
free from remarkable systemic or topical side effects, are suitable
for parenterally administering, preferably i.m. or s.c., active
ingredients which are hydrophilic or are made hydrophilic through
suitable derivatization, thereby obtaining a remarkable technical
improvement compared with the known technique.
[0020] A procedure has been found, consisting in the clinical,
post-mortem, and histological examinations of the animals treated
with the above mentioned microemulsions, suitable for thoroughly
evaluating the biocompatibility of any formulations for the
sustained release administration.
[0021] According to this procedure, a microemulsion is considered
acceptable, according to what stated in literature (Protein
Formulation and Delivery--F. J. McKelly 2000 pages 245-247), when
any local swelling, more or less marked depending on the
administered dose, is anyway reversible; on the other hand, a
similar tissular response is also observed for materials considered
biodegradable, which response is apparently important in affecting
the sustained release of the drug in time. A local intolerance in
the form of persistent ulcerations is conversely considered
unacceptable.
[0022] The microemulsions of the present invention consists for up
to 20% of an internal hydrophilic aqueous phase containing the
active ingredient, for 30 to 98% of a hydrophobic external phase
and for up to 50% of a surfactant alone or in admixture with a
co-surfactant. The microemulsion preferably contains a percentage
.ltoreq.35% of surfactant and co-surfactant, with a
surfactant/co-surfactant ratio .gtoreq.2, and most preferably
.gtoreq.3,5.
[0023] Other biologically compatible excipients which do not affect
the stability of the microemulsion can also be present.
[0024] Suitable surfactants for the microemulsions of the invention
are selected from natural or synthetic glycerophospholipids
containing residues of C.sub.4-C.sub.20 saturated or unsaturated
carboxylic acids, having as phosphoester moiety a residue of
choline, ethanolamine, serine, glycerol; cholesterol;
C.sub.12-C.sub.20 fatty acid esters of sugars such as sorbitol,
galactose, glucose, saccharose; polyoxyethylene sorbitan
C.sub.12-C.sub.20 fatty acid esters.
[0025] The optionally present co-surfactants are selected from
C.sub.8-C.sub.20 fatty acids, C.sub.2-C.sub.14 polyhydroxyalkanes,
particularly propylene glycol, hexanediol and glycerol,
C.sub.2-C.sub.12 alcohols, esters of lactic acid with a
C.sub.2-C.sub.8 alcohol residue.
[0026] The hydrophobic continuous phase is selected from the
following compounds, alone or in mixture: esters of
C.sub.8-C.sub.20 saturated or unsaturated carboxylic acids with a
C.sub.2-C.sub.8 alcohol residue or mono, di- and triglycerids of
C.sub.8-C.sub.20 fatty acids, or vegetable oils suitable for the
parenteral administration, such as soybean, peanut, sesame,
cottonseed, sunflower oils.
[0027] The microemulsions of the invention are further
characterized by pH ranging from 4.5 to 7.5, preferably from 5 to
7, said pH, when not intrinsic to the composition of the
microemulsion, being preferably obtained by addition of a suitable
amount of a natural amino acid to the microemulsion without
affecting its stability and the average size of the droplets.
[0028] The w/o microemulsions of the invention are particularly
suitable as carriers for peptides, in particular LHRH analogs such
as Leuprolide acetate, Goserelin, Triptorelin, Nafarelin acetate,
Histrelin, Cetrorelix or the corresponding acetates, or peptides
such as Somatostatin or its analogs such as Octreotide and
Lanreotide acetate.
[0029] Furthermore, the microemulsions of the invention are
particularly suitable as carriers for polysaccharides, in
particular unfractioned heparin or low molecular weight
heparins.
[0030] The microemulsions of the invention allow to prepare
formulations with sustained release of hydrophilic active
ingredients. Said sustained release formulations, which are a
further object of the invention, induce no local ulcerogenicity and
produce non persistent granulomas, which are reabsorbed during the
time in which the medicament is effective. In the case of LHRH-type
peptides, and in particular Leuprolide, Goserelin, Triptorelin,
Nafarelin acetate, Histrelin, Cetrorelix and the corresponding
acetates, sustained release for at least 30 days can be obtained.
In the case of Somatostatin, Octreotide and Lanreotide, sustained
release for at least 8 days can be obtained.
[0031] The invention further relates to the use of a microemulsion
according to the invention comprising Leuprolide, Goserelin,
Triptorelin, Nafarelin acetate, Histrelin, Cetrorelix or the
corresponding acetates for the preparation of a medicament for
suppressing testosterone production after single administration for
at least 30 days, where testosterone level already decreases 48 h
after the administration.
[0032] The invention also relates to the use of the microemulsions
containing Octreotide or its analogues for the preparation of a
medicament for suppressing growth hormone production for at least 8
days.
[0033] A further object of the present invention is the use of the
microemulsions containing unfractioned heparin or low molecular
weight heparins for the preparation of sustained release
medicaments upon single administration.
[0034] The following examples further illustrate the present
invention.
EXAMPLE 1
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0035] a) Preparation of the Aqueous Phase
[0036] 350 mg of Leuprolide acetate are dissolved in 10 ml of water
for injections added with 200 mg of lysine.
[0037] b) Preparation of the Oily Phase
[0038] 60 g of ethyl oleate, 25 g of soy lecithin (purity >95%)
and 5 g of caprylic acid are mixed separately, in a suitable vessel
thermostatized at a temperature of 60-70.degree. C., under
stirring. The resulting clear homogeneous solution is cooled at
room temperature.
[0039] c) Preparation of the Microemulsion
[0040] The aqueous phase (solution a) is added to the oily phase
(solution b) under stirring, to obtain an optically transparent,
homogeneous microemulsion. A pH value of about 6 was evaluated
based on the used amounts of lysine and caprylic acid fraction
soluble in the aqueous phase.
[0041] Said microemulsion is sterile filtered through a suitable
0.22 .mu.m membrane.
[0042] The Leuprolide acetate content of said microemulsion was
evaluated by HPLC analysis in the following conditions:
1 Stationary phase: Vydac C18 5 .mu. column (250 .times. 4 mm)
Mobile phase A: H.sub.20 + 0.1% TFA B: CH.sub.3OH + 0.1% TFA
Gradient: 20' 100% A to 100% B Flow: 0.8 ml/min Detector: UV 214
nm
[0043] The content in Leuprolide acetate is 3 mg/ml.
EXAMPLE 2
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0044] The procedure of Example 1 is followed, but solubilizing 600
mg of Leuprolide acetate in 10 ml of water.
EXAMPLE 3
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0045] The procedure of Example 1 is followed, but without adding
200 mg of lysine. Calculated pH is about 3.
EXAMPLE 4
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0046] The procedure of Example 1 is followed, but solubilizing 900
mg of Leuprolide acetate in 10 ml of water.
EXAMPLE 5
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0047] The procedure of Example 1 is followed, but changing the
amounts of surfactant and co-surfactant to 15 g of soy lecithin and
3 g of caprylic acid, respectively. In this way, although keeping
the ratio between the two components unchanged (5:1) the total
amount of the surfactant/co-surfactant mixture is changed from 30%
to 18%.
EXAMPLE 6
Preparation of a w/o Microemulsion Containing Octreotide
[0048] The procedure of Example 1 is followed, but solubilizing 3 g
of Octreotide in 10 ml of water, instead of 350 mg of Leuprolide
acetate in 10 ml of water.
EXAMPLE 7
Preparation of a w/o Microemulsion Containing Heparin
[0049] The procedure of Example 1 is followed, but solubilizing in
10 ml of water 50 mg of unfractioned heparin in the form of calcium
or sodium salt, instead of 350 mg of Leuprolide acetate.
EXAMPLE 8
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0050] The procedure of Example 1 is followed, but changing the
quali-quantitative composition of the oily phase: ethyl oleate
(66.9%), phosphatidyl choline (19.4%) and a 3:1 caproic
acid-butyric acid mixture (totally 3.9%).
EXAMPLE 9
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0051] a) Preparation of the Aqueous Phase
[0052] 60 mg of Leuprolide acetate are dissolved in 0.6 ml of water
for injections added with 8 mg of lysine.
[0053] b) Preparation of the Oily Phase
[0054] 2.1 g of ethyl oleate, 215 mg of polyoxyethylene sorbitan
monooleate and 1 g of soy lecithin are mixed in a suitable vessel,
heating at 50.degree. C. under stirring. The resulting clear
homogeneous mixture is cooled at room temperature.
[0055] The aqueous solution is slowly added in portions to the oily
mixture under stirring, to obtain an optically clear microemulsion
containing 1.5% of Leuprolide acetate.
EXAMPLE 10
Preparation of a w/o Microemulsion Containing Leuprolide
Acetate
[0056] a) Preparation of the Aqueous Phase
[0057] 60 mg of Leuprolide acetate are dissolved in 0.2 ml of water
for injections added with 4.1 mg of lysine.
[0058] b) Preparation of the Oily Phase
[0059] 1.2 g of ethyl oleate, 0.5 g of polyoxyethylene sorbitan
monooleate and 0.1 g of caprylic acid are mixed in a suitable
vessel, heating to 50.degree. C. under stirring. The resulting
clear homogeneous mixture is cooled at room temperature.
[0060] The aqueous solution is slowly added in portions to the oily
mixture under stirring, to obtain an optically clear microemulsion
containing 3 mg/ml of Leuprolide acetate.
EXAMPLE 11
Preparation of a w/o Microemulsion Containing Octreotide
Acetate
[0061] a) Preparation of the Aqueous Phase
[0062] 20 mg of Octreotide acetate are dissolved in 0.2 ml of water
for injections containing 0.2 g of propylene glycol and 4 mg of
lysine.
[0063] b) Preparation of the Oily Phase
[0064] 0.98 g of ethyl oleate, 0.5 g of soy lecithin and 0.1 g of
caprylic acid are mixed in a suitable vessel, heating to 50.degree.
C. under stirring. The resulting clear homogeneous mixture is
cooled at room temperature.
[0065] The aqueous solution is slowly added in portions to the oily
mixture under stirring. The resulting optically clear microemulsion
contains 10 mg/ml of Octreotide acetate.
EXAMPLE 12
Preparation of a w/o Microemulsion Containing Octreotide
Acetate
[0066] a) Preparation of the Aqueous Phase
[0067] 10 mg of Octreotide acetate are dissolved in 0.1 ml of water
for injections added with 2 mg of lysine.
[0068] b) Preparation of the Oily Phase
[0069] 0.59 g of ethyl oleate, 0.25 g of soy lecithin and 0.05 g of
caprylic acid are mixed in a suitable vessel, heating to 50.degree.
C. under stirring. The resulting clear homogeneous mixture is
cooled at room temperature.
[0070] The aqueous solution is slowly added in portions to the oily
mixture under stirring. The resulting optically clear microemulsion
contains 1% of Octreotide acetate.
EXAMPLE 13
Preparation of a w/o Microemulsion Containing Octreotide
Acetate
[0071] 0.59 g of ethyl oleate, 0.25 g of soy lecithin and 0.05 g of
caprylic acid are mixed in a suitable vessel, heating to 50.degree.
C. under stirring. The resulting clear homogeneous mixture is
cooled at room temperature. 0.1 g of water for injections
containing 2 mg of lysine, are gradually added under stirring to
the oily solution. The resulting optically clear microemulsion is
added under stirring with 10 mg of Octreotide acetate. The active
ingredient is inbodyrated in said microemulsion within a few
seconds. The microemulsion is filtered through a 0.22 mcm
polysulfone filter. The resulting optically clear microemulsion,
analyzed by HPLC, shows a content in Octreotide acetate of 6.35
mg/ml.
EXAMPLE 14
Preparation of a w/o Microemulsion Containing Myoglobin
[0072] a) Preparation of the Aqueous Phase
[0073] 5.5 mg of Myoglobin are dissolved in 1.5 ml of water for
injections added with 10 mg of lysine.
[0074] b) Preparation of the Oily Phase
[0075] 2 g of ethyl oleate, 1.2 g of soy lecithin and 0.25 g of
caprylic acid are mixed, in a suitable vessel, heating to
50.degree. C. The resulting clear homogeneous mixture is cooled at
room temperature.
[0076] The aqueous solution is slowly added in portions to the oily
mixture under stirring. The resulting optically clear microemulsion
contains 1.3 mg/ml of Myoglobin.
EXAMPLE 15
In vivo Evaluation of the Effectiveness of the Microemulsion
Containing Leuprolide Acetate Prepared as Described in Examples 1
and 2.
[0077] Three groups of Sprague Dawley male rats (10 per each group)
were housed for 5 days with water and food ad libitum before
treatment.
[0078] Two microemulsion formulations prepared as described in
Examples 1 and 2, with different concentrations of Leuprolide
acetate, namely 3 mg/ml (Example 1) and 6 mg/ml (Example 2) were
administered in a single dose, each in a group of rats, at the dose
of active corresponding to 0.750 mg/kg.
[0079] Control animals (3 per group) received saline solution.
[0080] Testosterone plasma levels after taking the blood samples
were evaluated at days 1, 2, 3, 4, 7, 14, 21, 28, 42 and 56. Blood
samples were centrifuged and serum testosterone was measured by EIA
kit.
[0081] Serum testosterone levels were evaluated until 60 days after
administration.
[0082] The results are described in FIG. 1.
[0083] The following Table 1 summarizes the data concerning organs
weight in the treated animals.
2TABLE 1 Effect of the microemulsions prepared as described in
Examples 1 and 2 on body and reproductive organs weight in rats.
Body and reproductive organs weight (g) after 28 and 56 days 28
days 56 days Seminal Seminal Microemulsion Body Testes Prostate
vesicles Body Testes Prostate vesicles Salina 570.0 .+-. 13.2 3.73
.+-. 0.09 0.72 .+-. 0.07 1.95 .+-. 0.010 631.2 .+-. 10.1 3.54 .+-.
0.05 0.80 .+-. 0.05 1.96 .+-. 0.26 Example 1 546.6 .+-. 8.8 1.84
.+-. 0.11* 0.33 .+-. 0.03* 0.49 .+-. 0.08* 600.0 .+-. 16.4 2.39
.+-. 0.17* 0.50 .+-. 0.03.sup.# 1.39 .+-. 0.14 Example 2 532.8 .+-.
4.6 1.68 .+-. 0.14* 0.22 .+-. 0.03* 0.25 .+-. 0.03* 626.6 .+-. 25.5
2.5 .+-. 0.2.sup..diamond. 0.51 .+-. 0.08.sup.# 1.43 .+-. 0.22 Data
expressed as mean .+-. standard error of the group (5 animals) *p
< 0.001; .sup..diamond.p < 0.01; .sup.#p < 0.05 vs. saline
(ANOVA test and Bonferroni .dagger. test for multiple
comparisons).
[0084] The formulations of the present invention, independently on
the concentration of the active in the microemulsion, are free from
substantial systemic or local side effects and provide the
sustained release of the active ingredient for a duration of at
least 30 days and therapeutic efficacy already 48 h after the
injection.
[0085] The efficacy is comparable to that obtainable when using the
commercial depot formulation "Enantone" based on bioerodible
polymers.
EXAMPLE 16
Evaluation of the Subcutaneous Tolerability of the
Microemulsions
[0086] The test for the evaluation of the subcutaneous tolerability
was effected by injecting groups of at least 9 rats with a single
administration of the microemulsions reported in the following
Table 2.
[0087] Clinical, post-mortem and histologic examinations were
carried out 48 h, 7 days and 14 days after the administration.
3 TABLE 2 Presence of swelling Presence of (Mean score*)
ulcerations Amount 14 14 Compound (mg) 48 h 7 days days 48 h 7 days
days Microemulsion 125 .mu.l 2 2 1 No No No A.sup.(1) Microemulsion
500 .mu.l 3 3 2.5 No No No A.sup.(1) Microemulsion 125 .mu.l 3 3 3
Yes Yes Yes B.sup.(2)
[0088] This test considers biologically compatible the formulations
which induce no persistent local ulcerations, whereas the presence
of swelling is a function of the amount of product injected and of
the rate of elimination of the ethyl oleate from subcutaneous
tissues, as described by Howard et al. in Int. J. Pharm. 16 (1983)
31-39.
EXAMPLE 17
In vivo Evaluation of the Effectiveness of the Microemulsion
Containing Octreotide Acetate
[0089] A dose corresponding to 6 mg/rat of microemulsion containing
Octreotide prepared as described in Example 11 was administered to
6 male rats weighing about 175-200 g. Plasma samples were taken
before the administration of the compound and 0.5 hours, 24 hours,
4, 6 and 8 days after treatment.
[0090] Octreotide was extracted from plasma and analyzed with
LC-MS-MS apparatus vs. a calibration curve. Plasma levels are
reported in the following table 3:
4 TABLE 3 Time of sampling 0.5 h 24 h 4 days 6 days 8 days
Octreotide 539 40.2 62.5 4.5 7.6 (ng/ml)
[0091] Significant Octreotide plasma levels were found until 8 days
after treatment.
EXAMPLE 18
Evaluation of the Dose/Efficacy Ratio of the Microemulsion
Containing Leuprolide Acetate
[0092] The formulation containing Leuprolide acetate, prepared as
described in Example 2, was administered to rats, in a single dose
of 2.25 mg/kg (Example). Control animals received a corresponding
dose of saline solution (Control).
[0093] Testosterone plasma levels on blood samples were evaluated
at days 0, 1, 2, 3, 5, 7, 14, 21, 28, 42 and 56. Blood samples were
centrifuged and serum testosterone was measured by an Elisa
kit.
[0094] Results are shown in FIG. 2.
[0095] The data concerning organs weights in treated animals are
summarized in the following Table 4.
[0096] Table 4: Effect of a Leuprolide acetate microemulsion or of
a saline solution on the body and reproductive organs weight in
rats (Example and Control).
5 Body and reproductive organs weight (g) on 56 day Body Testes
Prostate Seminal vesicles Control 645 .+-. 34 3.73 .+-. 0.16 0.82
.+-. 0.04 1.96 .+-. 0.09 1.94 .+-. 0.42 0.35 .+-. 0.14 0.55 .+-.
0.16 Example 608 .+-. 35 (p < 0.01) (p < 0.5) (p <
0.05)
[0097] Reduction of both testosterone plasma levels and
reproductive organs weight is clearly evidenced 56 days after the
administration of the microemulsion.
* * * * *